Published online May 31, 2006

3030–3043 Nucleic Acids Research, 2006, Vol. 34, No. 10 doi:10.1093/nar/gkl378 Dominant-negative Pes1 mutants inhibit ribosomal RNA processing and proliferation via incorporation into the PeBoW-complex Thomas Grimm, Michael Ho¨ lzel, Michaela Rohrmoser, Thomas Harasim, Anastassia Malamoussi, Anita Gruber-Eber, Elisabeth Kremmer1 and Dirk Eick*

Institute of Clinical Molecular Biology and Tumour Genetics, GSF Research Centre for Environment and Health, Marchioninistrasse 25, 81377 Munich, Germany and 1Institute of Molecular Immunology, GSF Research Centre for Environment and Health, Marchioninistrasse 25, 81377 Munich, Germany

Received February 27, 2006; Revised April 19, 2006; Accepted May 2, 2006 Downloaded from

ABSTRACT biogenesis is the major metabolic challenge of rapidly proliferating cells, particular in tumour cells, as it The nucleolar PeBoW-complex, consisting of Pes1, consumes up to 80% of the total energy. However, little is Bop1 and WDR12, is essential for cell proliferation known about the molecular mechanism that ensure the equi- http://nar.oxfordjournals.org/ and processing of ribosomal RNA in mammalian librium between cell division and cells. Here we have analysed the physical and required for balanced cell proliferation (1). Recently it has functional interactions of Pes1 deletion mutants become evident that ribosome synthesis is cell cycle con- with the PeBoW-complex. Pes1 mutants M1 and M5, trolled and sensitive to growth factor and nutrient signalling, with N- and C-terminal truncations, respectively, and likewise inhibited upon stress signals. Interestingly, sev- displayed a dominant-negative phenotype. Both eral pivotal regulators of cell cycle progression and senes- mutants showed nucleolar localization, blocked cence, such as p19ARF reside within the and are processing of the 36S/32S precursors to mature also involved in the control of ribosome biogenesis. More- by guest on November 16, 2015 over, nucleolar like nucleophosmin not only function 28S rRNA, inhibited cell proliferation, and induced in the maturation of , but are also implicated in the high p53 levels in proliferating, but not in resting control of the tumour suppressors p53 and p19ARF (2,3). cells. Mutant M1 and M5 proteins associated Noteworthy, dysfunction of nucleophosmin is frequently with large pre-ribosomal complexes and co- associated with acute myeloid leukaemia and heterozygous immunoprecipitated Bop1 and WDR12 proteins mice develop myelodysplastic syndromes (4,5). In conclu- indicating their proper incorporation into the sion, the recent years have unravelled remarkable links PeBoW-complex. We conclude that the dominant- between the nucleolus and cell cycle regulation, thus under- negative effect of the M1 and M5 mutants is lining the importance of the nucleolus far beyond the produc- mediated by the impaired function of the PeBoW- tion of ribosomes. complex. The nucleolus owns a particular ability in sensitizing cellu- lar stress after ultraviolet (UV) radiation of cells. Using micropore irradiation, Rubbi and Milner (6) demonstrated that large amounts of nuclear DNA damage failed to stabilize INTRODUCTION p53 unless the nucleolus was affected. In addition, forcing Eukaryotic ribosome biogenesis is a highly regulated, evolu- nucleolar disruption by anti-upstream binding factor (UBF) tionary conserved process in the nucleolus. A large precursor antibody microinjection (in the absence of DNA damage), ribosomal RNA (pre-rRNA) is transcribed by Pol I and rap- different chemotherapeutic drugs, or cre-mediated deletion idly packaged into the 90S ribonucleoprotein particle (90S of the Pol I specific transcription factor TIF-IA also caused pre-RNPs) containing ribosomal proteins, non-ribosomal pro- p53 stabilization and a p53-dependent cell cycle arrest. This teins and snoRNA-containing ribonucleoprotein particles suggests that the nucleolus is a stress sensor responsible for (snoRNPs). The 90S pre-RNPs are processed into intermedi- the maintenance of low levels of p53, which are automatic- ates, which finally give rise to mature 40S and 60S ribosomal ally elevated as soon as nucleolar function is impaired in subunits (1). response to stress (6,7).

*To whom correspondence should be addressed. Tel: ++49 89 7099 512; Fax: ++49 89 7099 500; Email: [email protected]

Ó 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2006, Vol. 34, No. 10 3031

How does nucleolar stress result in the activation of cell dominant-negative effect on rRNA processing and cell cycle check points? Several mechanisms have been proposed growth. Both mutants were incorporated into the PeBoW- that link ribosome biogenesis to the cell cycle machinery in complex and induced accumulation of p53. Our data suggests mammalian cells. A central player in all models is Mdm2, that essential cellular functions of Pes1 are mediated by its a p53-specific ubiquitin ligase. Disturbance of ribosome bio- incorporation into the PeBoW-complex. genesis may decrease the demand for ribosomal proteins and thus lead to an excess of free ribosomal proteins as L5, L11 and L23, which directly bind and inactivate Mdm2 resulting MATERIALS AND METHODS in the accumulation of p53 (8–12). Alternatively, export of ribosomal subunits to the cytoplasm may be a critical step Cloning/plasmids for p53 degradation, which does not take place, if rRNA pro- Pes1 was cloned from cDNA using the following pri- cessing is inhibited (13). However, other models cannot be mers: HsPescadillo fwd: 50-GCCACCATGGGAGGCCTT- excluded. GAGAAGAAG-30; HsPescadillo bwd: 50-CTCCGGCCTTG- We have recently described the nucleolar complex PeBoW, CCTTCTTGGCCTTC-30. Cloning into the modified pUC18 consisting of Pes1 (Pescadillo), Bop1 (block of proliferation) vector resulted in the addition of a C-terminal HA-tag to the and WDR12 (WD-repeat ) in mammalian cells. Pes1 open reading frame. The mutant Pes1-HA alleles were Knockdown of WDR12 by siRNA technology or expression created by standard techniques of molecular biology using of a dominant-negative WDR12 mutant blocked processing restriction enzymes and site directed mutagenesis. Pes1-HA Downloaded from of the 32S pre-rRNA, evoked stabilization of p53 and induced wild-type and mutants were cloned into the vector pRTS-1 a strong cell cycle arrest (14). Likewise, expression of using the SfiI restriction site (22). dominant-negative mutants of other members of the complex, Pes1 and Bop1, inhibit rRNA processing and cell cycle pro- Tissue culture gression (15,16). TGR-1 rat fibroblasts (provided by J. Sedivy, Brown Univer- http://nar.oxfordjournals.org/ The structure and function of the PeBoW-complex appears sity, Providence, RI) and U2OS osteosarcoma cells were cul- to be highly conserved throughout evolution. A homolog tured in DMEM with 10% FBS at 8% CO2. For generation complex consisting of Nop7 (Yph1p), Erb1 and Ytm1p has of polyclonal cell lines, 6 · 105 cells were transfected with been identified in yeast. As in , mutants of Nop7 7.5 mg pRTS-1 plasmids using Polyfect (QIAGEN) and selec- and Ytm1 inhibit rRNA processing and cell cycle progression ted in the presence of 200 mg/ml hygromycin B for 10 to (17). Mutations in Ytm1 disrupt interactions between Ytm1 14 days. Conditional expression was induced with 1 and Erb1, destabilize the heterotrimer, and significantly mg/ml doxycycline. The percentage of induced cells was reduce association of all three proteins with 66S pre-

monitored by FACS analysis for EGFP expression. by guest on November 16, 2015 ribosomes (18). Even though a function of the PeBoW-complex in pro- BrdU light assay cessing of the 32S rRNA precursor is established, the struc- ture of the complex and the role of its components Pes1, BrdU light assays were performed essentially as described Bop1 and WDR12 in other cellular processes is largely unre- previously (23). Briefly, stable polylclonal TGR-1 cells were seeded in the presence of 1 mg/ml doxycycline at a dens- solved e.g. mouse embryos lacking Pes1 arrest at morula 5 stages of development, their nucleoli fail to differentiate ity of 10 cells per 100 mm well. After 30 h seeding, the cells and accumulation of ribosomes is inhibited, suggesting an were incubated with 100 mM BrdU and doxycycline for 72 h. essential role of Pes1 for ribosome biogenesis and nucleolo- Culture medium was then removed and replaced by medium genesis (19). Overexpression of Pes1 can replace the SV40 T containing doxycycline and Hoechst 33 258 at 2 mg/ml for antigen in inducing colony formation in soft agar growth but 3 h. Finally, cells were placed on sheet of glass 11 cm not in inducing cell immortalization (20). Transient depletion above a 30 W fluorescent daylight bulb and irradiated from of Pes1 resulted in an increase of abnormal mitoses with beneath for 15 min. Cells were washed in phosphate-buffered appearance of binucleate or hyperploid cells, of cells with saline (PBS) two times and regular culture medium without multipolar spindles and of aberrant metaphase plates (21). doxycycline was added. Thus, Pes1 appears to be involved in multiple cellular pro- 32 in vivo cesses, yet it is unclear, whether all these processes require RNA analysis and P labelling the PeBoW-complex. Total RNA was isolated from TGR-1 cells using Trifast (Peq- Recently, the interaction of Pes1 transposon insertion Lab). A total of 2 mg of RNA were separated on a 1% mutants with Bop1 protein has been studied. Several of the agarose-formaldehyde gel and blotted on Hybond N+ mem- mutants displayed a dominant-negative phenotype for rRNA branes (GE Healthcare). The following 32P-labelled oligonuc- processing. Interestingly, the dominant-negative phenotype leotides were used to visualize rRNA precursors: ITS-1, 50- required the interaction of the mutant Pes1 with Bop1, CCGGAGAGATCACGTACCACCCCCGGTGCACACGA- while Pes1 mutants, which did not interact with Bop1, failed GATCACGGAGCCG-30;ITS-2,50-GGAGCGGTCGGCCC- to induce a dominant-negative phenotype (15), suggesting CGGTAGAGGGAGCGGGGGAGGAGAGGGACGCGAG- that Pes1 mutants might act only in the context of the 30; 18S, 50-CACCCGTGGTCACCATGGTAGGCACGGCG- PeBoW-complex. ACTACCATCGAAAGTTGATAG-30. Here we report the generation of a set of Pes1 deletion For metabolic labelling of rRNA, TGR-1 cells were mutants and their interaction with the PeBoW-complex. induced with doxycycline for 24 h, followed by pre- Two mutants with a N- and C-terminal deletion displayed a incubation in phosphate-free DMEM (Gibco) with dialyzed 3032 Nucleic Acids Research, 2006, Vol. 34, No. 10

FBS (Gibco) for 30 min. The medium was then replaced by acquisition software (Improvision) and a microscope (model phosphate-free DMEM/10% dialyzed FBS containing 15 mCi/ Axiovert 200 M; Carl Zeiss MicroImaging, Inc.) with ml 32P-orthophosphate (Amersham). After 60 min the radio- 63 (1.15) and 100 (1.30) plan oil objectives connected to a active medium was removed and cells were incubated in five charge-coupled device camera (model ORCA- 479; regular DMEM/10% FBS for indicated times. A total of Hamamatsu). 2 mg of RNA were separated on 1% agarose-formaldehyde gels. After electrophoresis, gels were placed on whatman- Immunoprecipitation paper and dried at 80C under vacuum suction. Dried agarose For immunoprecipitations, U2OS cells were seeded at sub- gels were exposed to regular X-ray films (Kodak) and rRNA confluent density and treated with 1 g/ml doxycycline for was visualized by autoradiography. A PhosphoImager (Fuji) m 20 h. Cells were harvested by trypsination and washing was used for the quantification of signal intensities. three times with PBS. For lysis, cells were resuspended in Sucrose gradients lysis buffer [50 mM Tris–HCl (pH 8.0), 1% NP-40, 150 mM NaCl, protease inhibitors, phosphatase inhibitors] and Sucrose solutions were prepared in 50 mM Tris–HCl incubated on ice for 20 min. After centrifugation, protein (pH 8.0), 20 mM NaCl. Sucrose gradients from 50–5% G beads (incubated with antibodies for at least 1 h, washed were prepared on top of a 300 ml CsCl (1.4 g/ml) cushion. twice after incubation) were incubated with the lysate over 7 A total of 1 · 10 cells, without pre-treatment with cyclohex- night at 4 C. The beads were washed three times with lysis Downloaded from emide, were lysed in 1 ml lysis buffer [10 mM HEPES buffer (lacking phosphatase- and protease inhibitor) and (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.1% resuspended in SDS-loading buffer/lysis buffer 1:1 with the NP-40, protease inhibitors] at 4C for 5 min. A total of 20 mg volume equal to the volume of cell lysate used for the IP. of glass beads were added each and total cell lysates were sonificated 10 times for 1 s. A total of 100 ml of total cell lys- ate were loaded on top of the sucrose gradients. Sub- http://nar.oxfordjournals.org/ sequently, sucrose gradients were centrifuged at 30 000 RESULTS r.p.m. for 2.5 h using a Ti60W rotor. Fractions of 200 ml Cloning and conditional expression of human each were collected. A total of 20 ml of each fraction were Pes1 mutants used for SDS–PAGE electrophoresis. The Pes1 protein is highly conserved in eukaryotes and sev- siRNA transfection eral domains and motifs have been predicted based on 4 5 sequence analysis (Figure 1A). Pes1 contains an evolutionary The day before transfection, 5 · 10 to 10 U2OS cells were highly conserved N-terminal pescadillo-like protein domain seeded in 6-well plates. A total of 5 mlof20mM control or (NPLP-domain, -database PF06732) at the N-terminus, by guest on November 16, 2015 Pes1-specific siRNA were diluted in 150 ml Optimem (Invit- a BRCT domain located in the middle of the protein, three rogen). A total of 150 ml Optimem containing 5 ml Oligo- classical nuclear localization sequences (NLS) distributed fectamine (Invitrogen) was added and incubated for 15 min. over the protein, six bipartite NLS at the C-terminus and Finally, 600 ml Optimem was added and applied to cells after two stretches of acidic amino acids near the C-terminus aspiration of the culture medium. Cells were incubated for (24). Pes1 is modified by SUMOylation, but SUMOylation 6 h. The following sequences (sense) were used: Pes1, CCA- at the consensus site, yKXE, located at the C-terminus has GAGGACCUAAGUGUGAdTdT; Control (nonspecific yet not been experimentally confirmed (25). Human and siRNA), UUCUCCGAACGUGUCACGUdTdT. mouse Pes1 proteins are 89% identical (Figure 1A). Systematic deletions are an appropriate tool for the invest- Immunoblotting and immunofluorescence igation of domains involved in subcelluar localization and Cells were directly lysed with 2· SDS-loading buffer (100 mM interactions as well as the generation of dominant-negative Tris–HCl, 200 mM DTE, 4% SDS, 10 mM EDTA, 0.2% mutants. A panel of deletion mutants covering the entire bromphenol blue, 20% glycerol). Cell lysates were separated open reading frame and a point mutation of the lysine in the by SDS–PAGE and blotted on nitrocellulose membranes (GE consensus SUMOylation site were constructed and tagged Healthcare). Equal loading of samples was controlled by Pon- with the hemagglutinin (HA) epitope at the C-terminus ceau S staining. Immunodetection was performed with anti- (Figure 1A). For the conditional expression in mammalian HA (3F10; Roche), anti-WDR12 (1B8), anti-Bop1 (6H11), cells, wild-type (wt) and mutant pes1 alleles were cloned anti-Pes1 (8E9) [Holzel et al. (14)], and anti-p53 (Pab-240; into the pRTS-1 vector (Figure 1B) (14,22). Dianova) antibodies. The expression of the gene of interest is tightly controlled For immunofluorescence, cells were grown on cover slides, by a doxycycline-regulable trans-activator and trans- fixed with ice cold methanol or methanol–aceton 1:1 and air repressor. Thus, this tight control prevents any selection dried. Unspecific binding was blocked with PBS/10% FBS. against vectors carrying dominant-negative pes1 alleles dur- p53 and HA-tagged Pes1 were detected with a 1:100 dilution ing normal tissue culture due to leakiness of the promoter. of anti-p53 antibody (Pab122; Dianova) and a 1:1000 dilution Moreover, the pRTS-1 vector allows for high expression of 3F10 anti-HA antibody, respectively. Primary antibodies levels of the gene of interest. In stably transfected polyclonal were incubated overnight at 4C in a humidified chamber. cell cultures, conditional gene activation is achieved in >95% Cy3 labelled secondary antibodies (Dianova) were incubated of cells (Figure 1C). As the bidirectional promoter of the for 1 h at RT. Nuclei were counterstained with DAPI (Sigma– pRTS-1 vector accomplishes simultaneous expression of Aldrich). Digital images were acquired using the Openlab EGFP in addition to the gene of interest, conditional gene Nucleic Acids Research, 2006, Vol. 34, No. 10 3033 Downloaded from http://nar.oxfordjournals.org/ by guest on November 16, 2015

Figure 1. Pes1 mutagenesis and conditional Pes1 expression. (A) A panel of Pes1 deletion mutants (M1–M8) were constructed. Recombinant Pes1 wt and mutant proteins carry a C-terminal HA-tag. NLS: classical nuclear localization sequence; the N-terminal NLS consists of three overlapping NLS; bip. NLS: six overlapping bipartite NLS; BRCT: BRCT domain; acidic domains: two glutamic acid rich regions; YKXE: consensus SUMOylation site; HA: hemagglutinin- tag. Mutant M8 has replaced Lys517 by Arg. (B) Pes1 and its mutants were expressed in rat fibroblasts (TGR-1) stably transfected with the inducible EBV-based vector pRTS-1. pRTS features a bidirectional promoter expressing simultaneously the gene of interest and EGFP. After induction by doxycycline, the vector switches from active silencing to transactivation. (C) EGFP expression was induced in >95% of cells 24 h after addition of doxycycline, as determined by FACS analysis. (D) Western blot analysis of wt or mutant Pes1 proteins in TGR-1 fibroblasts by anti-HA antibodies (3F10). The mock cell line expresses luciferase instead of Pes1-HA. The indicated cell lines were treated with 1 mg/ml doxycycline for 30 h (+) or left untreated (). Bottom panel shows Ponceau S staining as a loading control. 3034 Nucleic Acids Research, 2006, Vol. 34, No. 10 activation can be easily monitored by FACS analysis or fluor- localization and also some nucleoplasmic staining in cells escence microscopy (Figure 1B and C). Pes wt and mutants with high expression of Pes1 (Figure 2, wt). The deletion of M1–M8 were conditionally expressed in the rat fibroblast 54 N-terminal amino acids in mutant M1 did not affect the cell line TGR-1. Expression levels were determined 30 h nucleolar localization whereas the extension of this deletion after addition of doxycycline by western blot analysis. Most to amino acid 154 (M2) or 249 (M3) results in a diffuse nuc- of the mutant forms were detected at the expected size, how- lear staining. Deletion of the central part of Pes1 containing ever deletion of the acidic regions in M5 and M6 significantly the BRCT domain and adjacent uncharacterized regions in enhanced migration (Figure 1D). mutant M4 leads to a diffuse nucleoplasmic staining but with nucleolar staining in very few cells (Figure 2, data not shown). The C-terminal deletion mutants M5 to M7, together Subcellular localization of Pes1 mutants with mutant M8 harbouring a mutated consensus SUMOyla- Pes1 protein has been reported to localize predominantly to tion site, located to the nucleolus as Pes1. Therefore, our the nucleolus. To determine the subcellular localization of mutational analysis suggests that the region between 55 and the Pes1 mutants, we performed indirect immunofluorescence 154 amino acid plays an important role for the nucleolar loc- using HA-tag specific antibodies. As expected, the recombin- alization of the Pes1 protein. A contribution of the region ant Pes1 wt protein showed predominantly nucleolar extending from amino acid 155 to 249 remains elusive for Downloaded from http://nar.oxfordjournals.org/ by guest on November 16, 2015

Figure 2. Cellular localization of wt and mutant Pes1 proteins by indirect immunofluorescence. TGR-1 cells transfected with the indicated constructs were fixed with methanol/acetone after induction of the Pes1-HA alleles with doxycycline for 30 h. The HA-tagged proteins were stained by anti-HA antibodies (3F10), the nuclei were counterstained with DAPI. Nucleic Acids Research, 2006, Vol. 34, No. 10 3035

nucleolar localization in this study. Noteworthy, none of the mutants showed cytoplasmic staining suggesting that neither of the deletions affected the nuclear transport of Pes1. Since all mutants retained at least two putative NLS, the functional- ity of a single NLS could not be addressed by this set of mutants. N-terminal (M1) and C-terminal (M5) truncation mutants potently inhibit proliferation and trigger a reversible cell cycle arrest Next, we tested the effect of Pes1 deletion mutants on cell proliferation. Equal numbers of cells were seeded and expres- sion of wt Pes1, mutants M1 to M8 and luciferase (mock) was induced by addition of doxycycline. Cell numbers were determined after 6d (Figure 3A). Overexpression of Pes1 wt reduced the cell count to 62% compared to mock cells. A likewise decrease was observed for the mutants M6, M7 Downloaded from and M8, and to a higher extent for the mutants M2, M3 and M4. Expression of the mutants M1 and M5 resulted in the strongest reduction, 10 and 22% of mock cell count, respectively. FACS analysis revealed a significant increase of cells in the G0/G1 phase of the cell cycle 48 h after expres-

sion of mutants M1 and M5 (Table 1, Supplementary http://nar.oxfordjournals.org/ Figure S1). Hence, the mutants M1 and M5 suppressed cell proliferation in rat fibroblasts most efficiently. Further, we studied the ability of mutants M1 and M5 to mediate a reversible cell cycle arrest in a BrdU/light assay. In brief, dividing cells that incorporate BrdU into their DNA become highly photosensitive if they are additionally labelled with the Hoechst dye 33 258 and undergo apoptosis upon irradiation with visible light. Cell cycle arrested cells are protected from increased photosensitivity and survive by guest on November 16, 2015 BrdU/light treatment. Thus, cells that were arrested by condi- tional expression of an anti-proliferative Pes1 mutant, would subsequently give rise to colonies after withdrawal of doxy- cycline. Mock cells expressing luciferase showed only little colony outgrowth and thus demonstrating the low background of this assay. Expression of Pes1 wt did not increase the num- ber of colonies in comparison to the mock situation. The Pes1 mutant M1 provoked a pronounced rescue effect and sub- sequent colony outgrowth, consistent with a strong reversible cell cycle inhibition. The effect of mutant M5 was less intense but still significant. Mutant M3 showed a colony num- ber slightly over the background level, while all other Pes1 mutants remained at the background level. The reduced pro- liferation rates of Pes1 wt (62% in the proliferation assay) and mutants M2–M4 and M6–M8 is not accompanied by an Figure 3. Pes1 mutants M1 and M5 inhibit cell proliferation and elicit increased apoptosis rate (data not shown) or altered cell a reversible cell cycle arrest. (A) Equal numbers of TGR-1 cells stably cycle distribution (Table 1), thus the reason for the reduced transfected with the indicated constructs were seeded in multiples in the presence of 1 mg/ml doxycycline. Cells were trypsinized and cell count remains currently unclear. Taken together, the counted by trypan blue exclusion after 6d. The histogram depicts the mutants M1 and M5 mediated a potent inhibitory effect on cell counts relative to the mock cell line after 6d. Error bars indicate SD. cell proliferation and triggered a reversible G0/G1-specific (B) Reversible cell cycle arrest by overexpression of mutants M1 and M5. cell cycle arrest (Table 1). Stably transfected TGR-1 cell lines were seeded at low density and treated with 1 mg/ml doxycycline for 30 h to induce expression of The Pes1 mutants M1 and M5 inhibit the specified constructs. Subsequently, the cells were incubated with 100 mM BrdU for 72 h to label proliferating cells. Visible light pre-rRNA processing irradiation in the presence of Hoechst 33 258 selectively kills cells that Pes1 is involved in ribosome biogenesis and therefore we have incorporated BrdU in their DNA. Arrested cells survive BrdU light treatment and give rise to colonies after withdrawal of doxycycline. tested the ability of mutants M1 and M5 to inhibit maturation Images show representative BrdU light assays conducted with the indicated of ribosomal RNA. A short overview of the major mam- cell lines. malian rRNA processing pathway is depicted in Figure 4A. 3036 Nucleic Acids Research, 2006, Vol. 34, No. 10

Table 1. Cell cycle distribution of Pes1 mutants knockdown on ribosome biogenesis after in vivo labelling of rRNA. The production of the mature 28S rRNA was strongly Cell line G0/G1 %S%G2 % compromised (Figure 5B). Notably, synthesis of the large Mock 46.6 23.1 30.4 45/47S precursor was not affected, indicating that Pes1 is WT 47.7 24.6 27.7 not involved in rRNA transcription. In conclusion, expression M1 62.3 17.8 19.9 of dominant-negative Pes1 and depletion of endogenous Pes1 M2 43.7 26.5 29.9 block rRNA processing of the 32S rRNA precursor. M3 53.5 18.2 28.3 M4 47.6 21.7 30.7 M5 59.1 18.9 21.9 Mutants M1 and M5 induce p53 accumulation M6 48.6 24.8 26.6 in proliferating, but not in starving cells M7 49.5 22.6 27.9 M8 46.7 24.8 28.5 As mentioned before, p53 is believed to induce cell cycle arrest following nucleolar stress. This raised the question if Stably transfected TGR-1 cells were induced at subconfluent density for 48 h the observed impact of mutants M1 and M5 on pre-rRNA with doxycycline and subjected to FACS analysis. The percentage of cells in different cell cycle phases are indicated. processing triggers the accumulation of p53. To address this question, we analysed p53 levels in TGR-1 cells 30 h after expression of wt and mutant Pes1 proteins by western blot- Pes1 wt and mutant forms were expressed in TGR-1 cells for

ting (Figure 6A). Pes1 wt and mock cells did not accumulate Downloaded from 30 h. Total RNA was isolated, and analysed by northern ana- p53 (Figure 6A, lanes 1–4). Expression of mutant M1 pro- lysis with hybridization probes specific for the internal voked a strong increase of p53 protein (lanes 5 and 6). A transcribed spacer 1 and 2 (ITS-1 and ITS-2) of the ribosomal less pronounced accumulation was seen for the mutants M3 pre-RNA (Figure 4A). Expression of M1 and M5-induced a and M5 (lanes 9, 10 and 13, 14), whereas all other mutants significant accumulation of the 36S precursor as visualized caused no change in p53 levels. If disturbance in ribosome by hybridization with the ITS-1 specific probes (Supplement- http://nar.oxfordjournals.org/ biogenesis is the reason for p53 accumulation, then the accu- ary Figure S2). Inhibition of pre-rRNA processing becomes mulation of p53 should be diminished in cells in the absence even more pronounced, if rRNAs were hybridized with the of active ribosome biogenesis, such as serum-starved cells. ITS-2 specific probe. Mutants M1 and M5 lead to a strong To test this assumption, subconfluent TGR-1 cells were cul- increase of the amount of 32S rRNA precursor (Figure 4B, tured with 0.1% FBS for 72 h prior to the addition of doxy- upper arrows). While both mutants, M1 and M5, induced cycline. The cells were lysed 30 h later. Serum starvation did accumulation of the 36S/32S rRNA, their effect on pro- not affect the induction rate of recombinant proteins cessing of the 12S rRNA intermediate differed. The level (Figure 6B, lower panel, see also Figure 1D), however, of 12S pre-rRNA was reduced for the mutant M1, but

neither of the Pes1 mutants was able to trigger the accumula- by guest on November 16, 2015 appeared unaffected for the mutant M5 (Figure 4B, lower tion of p53 (Figure 6B, upper panel). arrows). This may indicate separable and common functions To further investigate the p53 accumulation induced by the of the mutants M1 and M5 in rRNA processing. mutant Pes1 proteins on the single cell level, we analysed p53 In addition we studied the impact of mutants M1 and M5 accumulation by immunofluorescence (Figure 6C). In prolif- on rRNA processing in TGR-1 cells by metabolic erating cells expressing Pes1 wt, 8.9% of nuclei stained pos- 32P-labelling (Figure 4C). The production of mature 28S itive for p53. Expression of Pes1 mutants M1 and M5 rRNA was severely reduced resulting from an inefficient pro- increased the number of positive cells to 82.0 and 53.4%, cessing of the 32S rRNA precursor, as concluded from the respectively (Figure 6D). Serum starvation reduced the num- relative high abundance of metabolically labelled 32S ber of p53-positive cells for Pes1 wt to 0.71%, and for the rRNA (Figure 4D). In contrast, synthesis of mature 18S mutants M1 and M5 to 8.7 and 3.3%, respectively. Thus rRNA was almost unaffected. These results are in line with neither of the mutants were able to induce p53 significantly our northern blot analysis. In conclusion, the deletion of the in serum-starved cells supporting the notion, that ribosome N-terminus or the C-terminus of Pes1 either compromises biogenesis is a prerequisite for the dominant-negative action ribosome biogenesis by blocking rRNA processing at the of Pes1 mutants M1 and M5. level of the 32S rRNA precursor. As previously demonstrated, N-terminally deleted WDR12 elicited a p53-dependent cell cycle arrest that was attenuated Endogenous Pes1 is required for rRNA processing by the coexpression of the human papillomavirus protein E6 and cell proliferation (14), a ubiquitin ligase targeting p53 for degradation. In line with these studies, mutants M1 and M5-induced cell cycle To confirm the role of Pes1 in ribosome biogenesis and cell arrest was alleviated in TGR-1 cells stably transfected with proliferation, we performed small interfering RNA (siRNA) HA-tagged E6 (Supplementary Figure S3). knockdown experiments of endogenous Pes1. We used human osteosarcoma U2OS cells, because we had generated a monoclonal antibody directed against human Pes1 (14). Mutants M1 and M5 associate with the large Cells were transfected at day 0, 1 and 2 with either control pre-ribosomal subunit or Pes1-specific siRNA. Expression of endogenous Pes1 The association of Pes1 mutants with pre-ribosomal complexes was dramatically reduced, as monitored by western blot ana- was studied by sucrose gradient fractionation using TGR-1 lysis 2d after the last transfection (Figure 5A). Moreover, pro- total cell lysates (Figure 7). All recombinant proteins accumu- liferation of Pes1-depleted cells was severely impaired (data lated in low molecular fractions, most likely due to an access of not shown). Further, we investigated the impact of Pes1 overexpressed free protein in total cell lysates. However, Pes1, Nucleic Acids Research, 2006, Vol. 34, No. 10 3037 Downloaded from http://nar.oxfordjournals.org/ by guest on November 16, 2015

Figure 4. Dominant-negative Pes1 mutants inhibit pre-rRNA processing. (A) Schematic of eukaryotic pre-rRNA processing. The top diagram shows the primary 47S transcript at the correct scale and the position of the probes used in the northern blots. The lower diagram shows the processing of the pre-rRNA schematically. The effects of M1 and M5 overexpression are indicated. (B) Northern blot for pre-rRNA probed with ITS-2. Total RNA was extracted from subconfluent TGR-1 cells transfected with the indicated constructs after 30 h of induction. Hybridization with a probe against the 18S rRNA confirms equal loading of the blot. (C) Expression of dominant-negative Pes1 mutants impairs formation of mature 28S rRNA. Asynchronously growing TGR-1 cells were treated with doxycycline for 24 h. Cells were pulse labelled with 32P-orthophosphate for 1 h and chased in regular medium for 0.5 and 3 h. (D) Ratio of 32S/28S rRNA at 3 h after metabolic labelling with 32P-orthophosphate as described in (C). and mutants M1 and M5, but not the mutant M3, exhibited spe- Incorporation of mutant Pes1 proteins into cific enrichment in high molecular weight fractions that co- the PeBoW-complex fractionated with ribosomal particles. Given the fact that M1 and M5 impair rRNA processing at the level of the 32S pre- Previously we showed that Pes1 forms a stable complex with rRNA and co-sediment in fractions similar to the 60S peak two other nucleolar proteins, Bop1 and WDR12, respectively (see 28S RNA), our data suggests that mutants M1 and M5 (PeBoW-complex). Interestingly, several of the deletion accumulate with the 66S pre-ribosomal large subunit. mutants generated in this study localized to the nucleolus 3038 Nucleic Acids Research, 2006, Vol. 34, No. 10

revealed no unspecific reactivity of the antibodies (Figure 8A). The endogenous PeBoW-complex was precipit- ated in IPs against WDR12 and Bop1, as expected (Figure 8A, lanes 3 and 4). HA-tagged Pes1 wt protein co-immunoprecipitated high amounts of WDR12 and Bop1 indicating a proper incorporation into the PeBoW-complex (Figure 8B, lane 2). The IPs against WDR12 and Bop1 could co-precipitate only a small fraction of HA-tagged Pes1 protein (Figure 8B, lanes 3 and 4). This might be due to the fact that 20 h after addition of doxycycline not all PeBoW-complexes have incorporated the HA-tagged Pes1 protein. Hence, the co-immunopreciptation of WDR12 and Bop1 with the anti-HA antibody is the most reliable readout for the incorporation of HA-tagged Pes1 proteins in the BeBoW-complex. The dominant-negative mutant M1 co-precipitated WDR12 and Bop1 proteins (Figure 8C, lane 2). Mutant M1 was also detectable in IPs against WDR12 and Bop1. This demon- Downloaded from strates that the Pes1 mutant M1 is efficiently incorporated into the PeBoW-complex (Figure 8C, lane 3 and 4). The mutant proteins M2 and M3 are not incorporated into PeBoW. Both proteins did not co-precipitate WDR12 and

Bop1 (Figure 8D and E, lane 2) and could not be precipitated http://nar.oxfordjournals.org/ in IPs against WDR12 and Bop1 (Figure 8D and E, lane 3 and 4). This finding is compatible with a previous report, that the region in Pes1 needed for nucleolar localization is also needed for binding of Bop1 (15). Pes1 mutant M4 is also Figure 5. Endogenous Pes1 is required for rRNA processing. (A) U2OS cells not incorporated into the PeBoW-complex according to the were transfected at day 0, 1 and 2 with either control or Pes1-specific siRNA. failure to co-precipitate significant amounts of WDR12 and Endogenous Pes1 levels were analysed by western blot analysis 2d after the Bop1 (Figure 8F, lane 2). The dominant-negative mutant M5, last transfection. Tubulin is shown as a loading control. (B) U2OS cells were similar to Pes1 wt and mutant M1 protein, co-precipitates transfected only twice at day 0 and 1 and metabolically labelled with by guest on November 16, 2015 32P-orthophosphate for 60 min at day 3. Subsequently, cells were incubated WDR12 and Bop1 (Figure 8G, lane 2) and is co-precipitated for 3 h in regular culture medium. Labelled rRNAs are indicated. by WDR12 and Bop1 (Figure 8G, lane 3 and 4). Mutants M6, M7 and M8 are all incorporated into the PeBoW-complex, as with or without a dominant-negative phenotype. Thus we they co-precipitate WDR12 and Bop1 (Figure 8H–J, lane 2) asked, if these mutants are nucleolar upon incorporation and in reciprocal IPs (Figure 8H–J, lanes 3 and 4). Taken into the PeBoW-complex or if nucleolar localization is a fea- together, our immunoprecipitation studies revealed that all ture of Pes1 independent of PeBoW-association. Further, it mutants are efficiently incorporated in the PeBoW-complex was unclear if the Pes1 mutants interact with Bop1 or except the mutants M2, M3 and M4. Notably, the mutants WDR12 in conjunction or independently of each other. More- M2, M3 and M4 are the only mutants showing no proper nuc- over, analysing the incorporation of the dominant-negative leolar localization, suggesting that the nucleolar localization mutant proteins M1 and M5 into the PeBoW-complex of Pes1 is linked to the PeBoW-association. Interestingly, would help to unravel the mechanism of their inhibitory func- we observed no independent interaction of Pes1 mutants tion. To address these issues, we performed a series of vice- with either Bop1 or WDR12. Further, our results propose versa immunoprecipitation experiments. As our antibodies that dominant-negative forms of Pes1 require incorporation against the PeBoW components WDR12 and Bop1 are into the PeBoW-complex for their negative effect on human specific, we performed the experiments in the ribosomal rRNA processing and cell cycle progression. human osteosacroma cell line U2OS. This cell line also allowed proper conditional expression of Pes1 mutants by the pRTS-1 vector. U2OS cells were plated at a subconfluent density and the DISCUSSION Pes1 constructs were induced for 20 h with doxycycline. In mammals, Pes1, Bop1 and WDR12 proteins are compon- The immunoprecipitation experiments were carried out with ents of the evolutionary highly conserved PeBoW-complex, antibodies against the HA-tag, WDR12 and Bop1, as well which is required for maturation of the 60S ribosomal subunit. as an isotype control. Western blot analysis was then per- Although PeBoW is highly abundant in the nucleolus, the way formed with a HA-tag specific monoclonal antibody (3F10) and the place of its assembly is unclear. A homologous com- to detect the recombinant Pes1 protein and monoclonal anti- plex in yeast, composed of Nop7, Erb1 and Ytm1, associates bodies against WDR12 and Bop1 to detect the association of with four consecutive 66S pre-ribosomal subunits containing the recombinant Pes1 protein with endogenous PeBoW mem- the 27SA2, 27SA3, 27SB and the 25.5S plus 7S pre-rRNAs bers. In the mock cell line, the lack of the HA-tag, WDR12 (18,26). Similar as in mammals, the yeast complex is required and Bop1 specific signals in the IP against the HA-tag for proper processing of the 27S pre-rRNA to the mature Nucleic Acids Research, 2006, Vol. 34, No. 10 3039 Downloaded from http://nar.oxfordjournals.org/ by guest on November 16, 2015

Figure 6. Dominant-negative Pes1 mutants induce p53 accumulation in proliferating cells but not in quiescent cells. (A) Western blot for endogenous p53 levels. Stably transfected TGR-1 cells were induced at subconfluent density for 30 h with doxycycline. Cell lysates were prepared and analysed by immunoblotting for p53 accumulation using anti-p53 antibodies (Pab-240). n.s.: non specific. (B) Upper panel: western blot as described in (A), but the cells had been serum-starved for 72 h before induction. Lower panel: immunoblot against the HA-tag to confirm the expression of recombinant wt and mutant Pes1. (C) Analysis of the endogenous p53 response to expression of Pes1 wt, and mutants M1 and M5 by indirect immunofluorescence. Asynchronously proliferating and serum-starved (72 h) cells were treated with doxycycline for 24 h to induce the Pes1 constructs. Cells were fixed in methanol and stained against p53 with anti-p53 (Pab122). Cell nuclei were counterstained with DAPI. Representative images are shown. (D) Percentage of p53-positive nuclei in TGR-1 cells upon induction of Pes1 wt, and the dominant-negative mutants M1 and M5. Proliferating and serum-starved cells were analysed by immunofluorescence as described in (C). Numbersof examined cells are indicated in brackets. Error bars indicate SD of the percentage of p53-positive nuclei cells.

25S rRNA. Recent evidence suggests that the assembly of the to the nucleolus and occurs only after association with the trimeric yeast complex occurs in the nucleolus, with assembly pre-ribosome and/or the assembly of WDR12 in the complex. of Nop7 and Erb1 into the pre-ribosomes prior to Ytm1 (18). Whether Nop7 and Erb1 assemble before binding to 66S pre- Nucleolar transport and assembly of Pes1 into ribosomes is not known. A stable complex of Nop7 and Erb1 the PeBoW-complex has not been demonstrated yet, neither in yeast, nor for the homologues Pes1 and Bop1 in mammalian cells. This is In this study we have constructed a set of deletion mutants to intriguing since the interaction of Pes1 and Bop1 can easily characterize the domains of Pes1 required for the nucleolar be demonstrated in GST-pull-down and yeast two hybrid localization and assembly into the PeBoW-complex. The experiments (15). This suggests that the interaction of endo- Pes1 protein has an evolutionary highly conserved N-terminal genous Pes1 and Bop1 may be inhibited during their passage domain of pescadillo-like proteins of 250 amino acids 3040 Nucleic Acids Research, 2006, Vol. 34, No. 10 Downloaded from

Figure 7. Dominant-negative mutants M1 and M5 associate with large pre-ribosomal particles. Western-blot analysis of sucrose gradient fractions of TGR-1 cells http://nar.oxfordjournals.org/ expressing Pes1-HA, and Pes1-HA mutants M1, M3 and M5 with an HA-specific antibody. RNA of each fraction was analysed by northern analysis with 28S rRNA specific probe (lower panel). followed by a distal BRCT domain (27). While the NPLP- (15). The failure of mutants M2 and M3 proteins to immuno- domain is present in all eukaryotes, the BRCT domain is precipitate complexes containing Bop1 and WDR12 supports missing in some simple organisms as Plasmodium fal- this notion. Anyway, incorporation of both mutant proteins ciparum. Three of our deletions mutants, M1, M2 and M3, into the PeBoW-complex cannot take place, since they do successively lacked parts of the NPLP-domain. The removal not localize to the nucleolus. In conclusion, the NPLP- of 54 N-terminal amino acids of Pes1 in mutant M1 resulted domain is critical for nucleolar localization and assembly of by guest on November 16, 2015 in a strong dominant-negative phenotype. Sucrose gradient the PeBoW-complex. centrifugation, co-immunoprecipitation experiments and nat- The Pes1 mutant M4 harbours a deletion of the BRCT ive gel-electrophoresis experiments (data not shown) revealed (BRCA1 C-terminal) domain that was first described as an proper incorporation of mutant M1 protein into the PeBoW- essential domain of the tumour suppressor function of the complex. Hence, the N-terminal 54 amino acids of the NPLP- BRCA1 protein. Similar repeat sequences have been identi- domain are neither required for the assembly of the PeBoW- fied in many proteins that function in DNA damage, repair complex nor for the transport of Pes1 to the nucleolus. It is and replication. Many BRCT-containing proteins have more likely that the N-terminal domain of Pes1 fulfils other phospho-peptide binding activity suggesting that BRCT essential tasks after the assembly of the complex by recruit- repeats might mediate phosphorylation-dependent protein– ment of further factors, or by catalysing specific steps in the protein interactions in processes that are central to cell maturation of the 32S pre-rRNA precursor. However, an cycle checkpoint and DNA repair functions. It is tempting enzymatic activity of the complex has not been demonstrated to speculate that the BRCT domain in Pes1 might fulfil sim- yet, neither in mammals nor in yeast. The mutants M2 and ilar tasks and links nucleolar processes to cell cycle control M3 lacked larger parts of the NPLP-domain. Proteins of and DNA synthesis. Interestingly, a temperature sensitive both mutants were still transported into the nucleus. A trans- mutant of yeast Nop7, a homologue of Pes1, can specifically port into the nucleolus, however, was inhibited. This is in line inhibit S phase entry in yeast cells (17). This points to addi- with the observation that mutant M2 and M3 proteins did not tional cell cycle control mechanisms by Pes1, which may act co-immunoprecipitate Bop1 and WDR12, because the independently of ribosome biogenesis. The mutant M4 pro- assembly of the PeBoW-complex in the nucleolus could not tein did not co-immunoprecipitate Bop1 and WDR12 from take place. The absence of a dominant-negative phenotype cellular lysates. This lack of interaction suggests that the of both mutants also supports the model that incorporation NPLP-domain in Pes1 is not sufficient for binding of Bop1 of Pes1 mutants into the PeBoW-complex is a prerequisite in the nucleoplasm, or that the interaction of nucleoplasmic for a dominant-negative phenotype. The region deleted in Pes1 and Bop1 is negatively controlled by a yet unknown M2 and M3 has been reported to interact directly with mechanism. Importantly, in addition to the Bop1 interacting Bop1 (15). The interaction domain with Bop1 has been iden- domain, the BRCT domain also plays an important role in tified in murine Pes1 after transposon insertion mutagenesis nucleolar localization. of 19 extra amino acids downstream of amino acid The second mutant with a strong dominant-negative 198 (Pes1-tn2), amino acid 202 (Pes1-tn11) and amino acid phenotype on cell proliferation and rRNA processing was 204 (Pes1-tn12) (Figure 9) in yeast two hybrid experiments M5. Similar to M1, mutant M5 protein localized to the Nucleic Acids Research, 2006, Vol. 34, No. 10 3041 Downloaded from http://nar.oxfordjournals.org/ by guest on November 16, 2015

Figure 8. Incorporation of Pes1 mutants into the PeBoW-complex. U2OS cells stably transfected with the indicated constructs (A–J) were seeded at subconfluent density and treated with doxycycline for 20 h. The lysates were subjected to immunoprecipitation with antibodies either against the HA-tag (3F10), WDR12 (1B8), Bop1 (6H11) or the isotype control coupled to protein G Sepharose beads. The amount of protein loaded in each lane resembles an equivalent amountof total lysate or 20% thereof for the input. * indicates cross-reactivity to Ig molecules. nucleolus and co-immunoprecipitated Bop1 and WDR12. 12S pre-rRNA were unchanged for the mutant M5. This sug- However, we noticed a specific difference in the processing gests an inhibition of 12S pre-rRNA processing by mutant of the 5.8S rRNA for mutants M1 and M5. While the amount M5 and an accumulation of this precursor, whereas 12S of 12S pre-rRNA was reduced for mutant M1, the levels of pre-rRNA processing proceeds in the presence of mutant M1. 3042 Nucleic Acids Research, 2006, Vol. 34, No. 10 Downloaded from http://nar.oxfordjournals.org/

Figure 9. Boundaries and domains important for Pes1 function and PeBoW formation. Pes1 deletions mutants of this work were compared with transposon by guest on November 16, 2015 insertion mutants published by Lapik et al. (15). Deletion mutants conferring a dominant-negative phenotype or affecting formation of PeBoW are indicated.

The data suggests that the PeBoW-complex is required for mechanisms have been proposed for p53 degradation: nucle- consecutive rRNA processing steps during maturation of olar sequestration of Mdm2, and the blockage of a postulated the 60S ribosomal subunit and that mutant M5 can block nucleolar route of p53 export for cytoplasmic degradation the processing of the 12S pre-rRNA in addition to the 32S (13,31). The Mdm2 sequestration model is supported by the pre-rRNA. Accordingly, the C-terminal domain of Pes1 is recent finding that several ribosomal proteins, L5, L11 and required for processing of the 32S and 12S pre-rRNA pre- L23, can bind and inhibit the function of Mdm2. Inhibition cursors, whereas the N-terminal domain appears to be dis- of ribosome biogenesis leads to accumulation of free L5, pensable for processing of the latter. L11 and L23 proteins, which bind to and inhibit Mdm2- mediated p53 ubiquitination and degradation. Inhibiton of Mdm2 restores p53-mediated transactivation, accumulation Role of the PeBoW-complex in cell cycle control of p21 protein levels, and induction of cell cycle arrest The impaired function of the PeBoW-complex evokes a p53 (5–9,27). The second model postulates a nucleolar export response in proliferating but not in serum-starved cells. All route for Mdm2–p53 complexes and subsequent cytoplasmic three components of the PeBoW-complex are encoded by degradation of p53, termed by Sherr and Weber (13) as Myc target and usually are not expressed in quiescent Mdm2–p53 complexes ‘riding the ribosome’. This model is cells (14,28,29). This suggests that expression of dominant- supported by the observation that p53 can be covalently negative mutants of Pes1 (this paper), or WDR12 (14) may linked to 5.8S rRNA, and associates with a subset of be not sufficient for the induction of p53, because they cannot ribosomes (32–34). Further support comes from the finding assemble into the PeBoW-complex and block its function. that inhibition of nuclear export leads to accumulation of However, a recent study showed that human WI-38 fibroblasts p53 in the nucleus. If p53 has to enter the nucleolus for the accumulated p53 under growth-restricting conditions, such as second model, or is loaded on ribosomal subunits during serum starvation or confluency (30). the passage through the nucleoplasm, is currently an open Whether the PeBoW-complex is directly involved in the question. Evidence for the presence of p53 in the nucleolus degradation of p53 remains unclear. Since dominant-negative has recently been shown following cell permeabilization, Pes1 mutants act only in the presence of ongoing ribosome where most soluble nucleoplasmic p53 was eliminated, but biogenesis it is conceivable that signalling of p53 degradation nuclear-bound p53 remained readily detectable in the nucleoli should be linked to this process. Two non-mutually exclusive (6). Accumulation of nucleolar p53 has also been observed Nucleic Acids Research, 2006, Vol. 34, No. 10 3043

after inhibiton of the proteasome (35). Alternatively, p53 14. Holzel,M., Rohrmoser,M., Schlee,M., Grimm,T., Harasim,T., associate with large pre-ribosomal subunits, if they leave Malamoussi,A., Gruber-Eber,A., Kremmer,E., Hiddemann,W., the nucleolus. Comparable to processing of the 27S pre- Bornkamm,G.W., Kremmer,E. and Eick,D. (2005) Mammalian WDR12 is a novel member of the Pes1-Bop1 complex and is required rRNA in yeast (25), processing of the 32S pre-rRNA in mam- for ribosome biogenesis and cell proliferation. J. Cell. Biol., 170, malian cells probably occurs at the transition of the large pre- 367–378. ribosomal subunit from the nucleolus to the nucleoplasm. At 15. Lapik,Y.R., Fernandes,C.J., Lau,L.F. and Pestov,D.G. (2004) Physical this moment, the functionality of the PeBoW-complex is and functional interaction between Pes1 and Bop1 in mammalian ribosome biogenesis. Mol. Cell, 15, 17–29. required. Successful processing of the 32S rRNA would 16. Strezoska,Z., Pestov,D.G. and Lau,L.F. (2002) Functional inactivation cause the release of the PeBoW-complex and loading of of the mouse nucleolar protein Bop1 inhibits multiple steps in Mdm2 and p53. A non-functional PeBoW-complex would pre-rRNA processing and blocks cell cycle progression. J. Biol. Chem., inhibit processing of the 32S pre-RNA and thereby nuclear 277, 29617–29625. export of p53/Mdm2. 17. Du,Y.C. and Stillman,B. (2002) Yph1p, an ORC-interacting protein: potential links between cell proliferation control, DNA replication, and ribosome biogenesis. Cell, 109, 835–848. 18. Miles,T.D., Jakovljevic,J., Horsey,E.W., Harnpicharnchai,P., Tang,L. and Woolford,J.L.,Jr (2005) Ytm1, Nop7, and Erb1 form a complex SUPPLEMENTARY DATA necessary for maturation of yeast 66S preribosomes. Mol. Cell. Biol., Supplementary Data are available at NAR Online. 25, 10419–10432.

19. Lerch-Gaggl,A., Haque,J., Li,J., Ning,G., Traktman,P. and Downloaded from Duncan,S.A. (2002) Pescadillo is essential for nucleolar assembly, ribosome biogenesis, and mammalian cell proliferation. J. Biol. Chem., ACKNOWLEDGEMENTS 277, 45347–45355. 20. Maiorana,A., Tu,X., Cheng,G. and Baserga,R. (2004) Role of This work was supported by the DFG (EI 216/8-1, SFB684, pescadillo in the transformation and immortalization of mammalian SFB-Transregio5). Funding to pay the Open Access publica- cells. Oncogene, 23, 7116–7124. tion charges for this article was provided by the DFG. 21. Killian,A., Le Meur,N., Sesboue,R., Bourguignon,J., Bougeard,G., http://nar.oxfordjournals.org/ Gautherot,J., Bastard,C., Frebourg,T. and Flaman,J.M. (2004) Conflict of interest statement. None declared. Inactivation of the RRB1-Pescadillo pathway involved in ribosome biogenesis induces chromosomal instability. Oncogene, 23, 8597–8602. 22. Bornkamm,G.W., Berens,C., Kuklik-Roos,C., Bechet,J.M., Laux,G., REFERENCES Bachl,J., Korndoerfer,M., Schlee,M., Holzel,M., Malamoussi,A. et al. (2005) Stringent doxycycline-dependent control of gene activities using 1. Warner,J.R. (1999) The economics of ribosome biosynthesis in yeast. an episomal one-vector system. Nucleic Acids Res., 33, e137. Trends Biochem. Sci., 24, 437–440. 23. Pestov,D.G. and Lau,L.F. (1994) Genetic selection of growth-inhibitory 2. Brooks,C.L. and Gu,W. (2006) p53 Ubiquitination: Mdm2 and beyond. sequences in mammalian cells. Proc. Natl Acad. Sci. USA, 91, Mol. Cell, 21, 307–315. 12549–12553. by guest on November 16, 2015 3. Latonen,L. and Laiho,M. (2005) Cellular UV damage responses— 24. Haque,J., Boger,S., Li,J. and Duncan,S.A. (2000) The murine Pes1 functions of tumor suppressor p53. Biochim. Biophys. Acta, 1755, 71–89. gene encodes a nuclear protein containing a BRCT domain. Genomics, 4. Falini,B., Mecucci,C., Tiacci,E., Alcalay,M., Rosati,R., Pasqualucci,L., 70, 201–210. La Starza,R., Diverio,D., Colombo,E., Santucci,A. et al. (2005) 25. Kinoshita,Y., Jarell,A.D., Flaman,J.M., Foltz,G., Schuster,J., Cytoplasmic nucleophosmin in acute myelogenous leukemia with a Sopher,B.L., Irvin,D.K., Kanning,K., Kornblum,H.I., Nelson,P.S. et al. normal karyotype. N. Engl. J. Med., 352, 254–266. (2001) Pescadillo, a novel cell cycle regulatory protein abnormally 5. Grisendi,S., Bernardi,R., Rossi,M., Cheng,K., Khandker,L., Manova,K. expressed in malignant cells. J. Biol. Chem., 276, 6656–6665. and Pandolfi,P.P. (2005) Role of nucleophosmin in embryonic 26. Dez,C. and Tollervey,D. (2004) Ribosome synthesis meets the cell development and tumorigenesis. Nature, 437, 147–153. cycle. Curr. Opin. Microbiol., 7, 631–637. 6. Rubbi,C.P. and Milner,J. (2003) Disruption of the nucleolus mediates 27. Staub,E., Fiziev,P., Rosenthal,A. and Hinzmann,B. (2004) Insights into stabilization of p53 in response to DNA damage and other stresses. the evolution of the nucleolus by an analysis of its protein domain EMBO J., 22, 6068–6077. repertoire. Bioessays, 26, 567–581. 7. Yuan,X., Zhou,Y., Casanova,E., Chai,M., Kiss,E., Grone,H.J., 28. Schlosser,I., Holzel,M., Murnseer,M., Burtscher,H., Weidle,U.H. and Schutz,G. and Grummt,I. (2005) Genetic inactivation of the Eick,D. (2003) A role for c-Myc in the regulation of ribosomal RNA transcription factor TIF-IA leads to nucleolar disruption, cell cycle processing. Nucleic Acids Res., 31, 6148–6156. arrest, and p53-mediated apoptosis. Mol. Cell, 19, 77–87. 29. Schlosser,I., Holzel,M., Hoffmann,R., Burtscher,H., Kohlhuber,F., 8. Dai,M.S. and Lu,H. (2004) Inhibition of MDM2-mediated p53 Schuhmacher,M., Chapman,R., Weidle,U.H. and Eick,D. (2005) ubiquitination and degradation by ribosomal protein L5. J. Biol. Chem., Dissection of transcriptional programmes in response to serum and 279, 44475–44482. c-Myc in a human B-cell line. Oncogene, 24, 520–524. 9. Dai,M.S., Zeng,S.X., Jin,Y., Sun,X.X., David,L. and Lu,H. (2004) 30. Bhat,K.P., Itahana,K., Jin,A. and Zhang,Y. (2004) Essential role of Ribosomal protein L23 activates p53 by inhibiting MDM2 function in ribosomal protein L11 in mediating growth inhibition-induced p53 response to ribosomal perturbation but not to translation inhibition. activation. EMBO J., 23, 2402–2412. Mol. Cell. Biol., 24, 7654–7668. 31. Tao,W. and Levine,A.J. (1999) Nucleocytoplasmic shuttling of 10. Jin,A., Itahana,K., O’Keefe,K. and Zhang,Y. (2004) Inhibition of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. HDM2 and activation of p53 by ribosomal protein L23. Mol. Cell. Proc. Natl Acad. Sci. USA, 96, 3077–3080. Biol., 24, 7669–7680. 32. Fontoura,B.M., Atienza,C.A., Sorokina,E.A., Morimoto,T. and 11. Lohrum,M.A., Ludwig,R.L., Kubbutat,M.H., Hanlon,M. and Carroll,R.B. (1997) Cytoplasmic p53 polypeptide is associated with Vousden,K.H. (2003) Regulation of HDM2 activity by the ribosomal ribosomes. Mol. Cell. Biol., 17, 3146–3154. protein L11. Cancer Cell, 3, 577–587. 33. Fontoura,B.M., Sorokina,E.A., David,E. and Carroll,R.B. (1992) p53 is 12. Zhang,Y., Wolf,G.W., Bhat,K., Jin,A., Allio,T., Burkhart,W.A. and covalently linked to 5.8S rRNA. Mol. Cell. Biol., 12, 5145–5151. Xiong,Y. (2003) Ribosomal protein L11 negatively regulates 34. Samad,A. and Carroll,R.B. (1991) The tumor suppressor p53 is bound oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress to RNA by a stable covalent linkage. Mol. Cell. Biol., 11, 1598–1606. checkpoint pathway. Mol. Cell. Biol., 23, 8902–8912. 35. Klibanov,S.A., O’Hagan,H.M. and Ljungman,M. (2001) Accumulation 13. Sherr,C.J. and Weber,J.D. (2000) The ARF/p53 pathway. Curr. Opin. of soluble and nucleolar-associated p53 proteins following cellular Genet. Dev., 10, 94–99. stress. J. Cell. Sci., 114, 1867–1873.